Severe pulmonary hypertension is a progressive and irreversible disease that often leads to mortality due to right heart failure. The pathogenesis of pulmonary hypertension involves proliferation of cells, including endothelial cells (intimal remodeling), in the vessel walls of pulmonary arteries resulting in remodeling and pulmonary hypertension. Several factors implicated in the remodeling process are driven by hypoxia. One mechanism by which cells respond to hypoxia is by stabilization of hypoxia-inducible transcription factors (HIFs), HIF-1alpha and HIF-2alpha. Independent studies suggest that both HIF-1alpha and HIF-2alpha are important mediators in PH. However, recent investigations have reported a marked increase in HIF-2alpha gene locus polymorphism in individuals adapting to high altitudes, suggesting a role of HIF-2alpha in PH. Mechanism by which HIF-2alpha influences pulmonary vascular remodeling and PH, independent of HIF-1alpha is not known. This proposal is focused on understanding the role of HIF-2alpha and its pathways in the pathogenesis of pulmonary hypertension. We have recently identified a new pathway, the HIF-2alpha-A2A receptor pathway, by which HIF-2alpha can promote endothelial proliferation, independent of HIF-1alpha. We hypothesize that hypoxia increases endothelial proliferation and vascular remodeling through a HIF-2alpha and A2A receptor-dependent mechanism and that this is an important step in the pathogenesis of PH. Additionally, we have also identified HIF-1alpha in smooth muscle cell proliferation, independent of HIF-2alpha. The proposed studies are divided into three aims. Aim 1 will elucidate mechanisms by which hypoxia and HIFs influence endothelial growth using cultured primary pulmonary endothelial cells as well as endothelial cells derived from hypertensive rats. The second aim will be used to test the hypothesis that inhibition or knockdown of HIFs, particularly HIF-2alpha will limit vascular remodeling and PA pressures in a rat model of PAH. The third aim will test whether inhibition or knockdown of A2A receptor, a downstream transcriptional target of HIF-2alpha, can mitigate remodeling and PA pressures in a rat model of PAH. The in vivo aims will investigate effects using both a prevention model and a rescue model. Results of the proposed research will help provide insights into hypoxic pathways that influence pulmonary vascular remodeling and offer alternative targets for treatment of PH.